The development of techniques of gene isolation by DNA-mediated gene transfer in cultured animal cells provides a powerful tool for the analysis of the genetics of cancer. Some human tumors contain genes which can oncogenically transform mouse fibroblasts of the established cell line NIH 3T3. Many of the human oncogenes detected by the NIH 3T3 transfection assay belong to the ras gene family. The human oncogene homologue to the Kirsten sarcoma virus oncogene (human c-K-ras) has been found activated in a broad variety of human tumor cell lines and primary tumors. We have isolated human sequences which span over 45 kilobase pair of the c-K-ras oncogene activated in two lung adenocarcinomas (PR310 and PR371) propagated into nude mice. Nucleotide sequence analysis in concert with DNA transfection experiments indicate that the PR371 oncogene has been activated by a single base change in the first coding exon which results in the incorporation of cysteine instead of glycine at position 12 of the predicted amino acid sequence. The PR310 oncogene, however, has been activated by a point mutation at codon 61 of the second coding exon which substitutes histidine for glutamine in the c-K-ras gene product. These results indicate that the activation of the c-K-ras oncogene in human lung adenocarcinomas can occur by different mutational events. We have developed a novel method to detect and characterize single base substitutions in transcribed eukaryotic genes that can be applied for the diagnostic screening of human tumors for the presence of mutant ras oncogenes. Using this method we have shown that the mutant c-k-ras allele is amplified and overexpressed relative to the normal allele in PR310 and PR371 human tumor cells. We are now studying the mechanisms which regulate the expression of the oncogene in human normal and tumor cells and in mouse and rat fibroblast transformants. (M)